Ordering and sample preparation

The first step of the order process is to provide your patient’s medical history. You can do this through Nucleus, our online portal for ordering tests and receiving test results. Nucleus also allows you to track where your patient’s sample is in each step of the process.

Our first step upon receiving the sample is verifying whether it meets the quality criteria required to generate a complete and accurate result. If the sample is blood or saliva, it will be run through an extraction robot to extract the DNA. We then perform a series of molecular biology techniques to prepare the sample DNA for analysis.

First, the DNA is sheared into fragments, each comprised of about 1,000 nucleotides. Next is the adapter ligation step, which involves adding a synthetic DNA sequence to both ends of each DNA fragment. These synthetic ends allow the DNA fragment to interact with the sequencing system, while also tagging the DNA to a specific patient sample. Finally, the adapted DNA fragments are copied multiple times in a process called enrichment, which produces enough of the necessary DNA for successful sequencing.

All of the methods used in this preparatory phase are tested and trusted methods of DNA sequencing preparation. A reference DNA sample is also prepared alongside the patient samples to serve as the control during sequencing. As the sequencing results for this reference DNA are known, it ensures that the results obtained from the patient samples are reliable.

Targeted sequencing with NGS technology

In general, targeted sequencing refers to all technologies where only a selected portion of the genome is sequenced. A common, targeted sequencing approach is whole exome sequencing (WES) where specific molecular probes are used to capture DNA from the coding regions of >20,000 human genes. The process does not typically include the non-coding regions (>98.5%) of the human genome. In the context of specific phenotype-driven gene panels, a more targeted set of genes can be selected for sequencing. In targeted sequencing, the patient’s sample is prepared by adding sample specific DNA adapters to the ends of the DNA fragments, capturing specific genomic regions by hybridization, and amplifying the sequencing libraries using PCR.

In the sequencing instrument, the sequencing library that has been prepared from the patient’s DNA is flushed through the sequencing flow cell. A flow cell is a fluidics device where the DNA fragments in the sequencing library are sequenced. Millions of DNA fragments attached to the flow cell surface form clusters of thousands of identical molecules. Each molecule on the flow cell is sequenced using sequencing-by-synthesis approach where fluorescently labelled nucleotides latch on to the ends of the growing DNA strands and images are captured. Sequencing proceeds in cycles where old labels are cleaved off, new labelled nucleotides are added and the imaging process is repeated. The images reveal all the bases and their order in each of the immobilized DNA fragments. The information content on the images is converted to raw data and extracted to text files.

Whole Exome Sequencing

Blueprint Genetics Whole Exome targets all protein-coding genes of the genome as well as a number of clinically relevant deep intronic variants. It has been developed to maximize diagnostic yields by generating high-quality and uniform sequencing data across the whole exome.

High-quality analysis
  • On average, 99.4% of base pairs in the target region are covered at a sequencing depth of at least 20x
  • Highly uniform sequencing depth across all target genes
  • Single nucleotide variants (SNV): Sens 99.65% and Spec >99.99%
  • Insertions, deletions and indels
  • 1-10 bps: Sens 96.94% and Spec >99.99%
  • 11-50 bps: Sens 99.07% and Spec >99.99%

Clinical Exome Sequencing

The Blueprint Genetics Clinical Exome Sequencing (CES) assay aims to cover all clinically relevant genomic targets in inherited diseases. It consists of >4000 genes, including all of our panel genes in addition to candidate genes not currently on the panels but curated by our clinical interpretation team based on data in the Clinical Genomic Database (CGD) and The Developmental Disorders Genotype-Phenotype Database (DDG2P). A number of candidate genes have also been included which are not present in CGD/DD2GP databases but where relevant publications are strongly suggestive of disease association and the gene has either high pLI (> 0.9) or high missense constraint (>3.0-4.0) in gnomAD have also been included. Our CES assay includes disease-causing, non-coding intronic variants associated with disease phenotypes, additional genomic targets for quality control of INDEL sensitivity, mitochondrial DNA, and pharmacogenetic targets.

The assay is based on high-quality, clinical-grade NGS that offers precise clinical diagnostic testing. All of our diagnostic panels and single genes are sectioned from the high-quality CES assay.

Blueprint Genetics utilizes high-quality custom capture technology and next-generation sequencing (NGS) methods to obtain deep and uniform data. All of our NGS technology is subjected to thorough quality control measures, after which raw sequence reads are transformed into variants by our proprietary bioinformatics pipeline.

High-quality analysis
  • An average of 99.86% of base pairs in the target region are covered at a sequencing depth of at least 20x
  • Highly uniform sequencing depth across all target genes
  • Single nucleotide variants (SNV): Sens 99.89 % and Spec >99.99%
  • Insertions, deletions and indels
  • 1-10 bps: Sens 96.9% and Spec >99.99%
  • 11-50 bps: Sens 99.13% and Spec >99.99%

Data analysis and interpretation

Sequencing creates a massive amount of raw data, which must undergo significant processing to become clinically relevant information. The proprietary automated bioinformatics pipeline developed and used by Blueprint Genetics produces accurate and reliable results. All of the software, equipment, and algorithms used are industry-standard in the field of molecular genetics, but specific parameters have been adjusted and other customizations have been made to improve data processing and analysis.

The first step in analyzing the results is quality filtering of the raw sequencing reads. This allows any ambiguous or unmappable reads to be trimmed from the data pool. Once the highest-quality reads have been isolated, they are aligned with the human reference genome so variant calling can begin. Variants are differences between the patient’s sequences and the reference sequence. Hundreds to thousands may be found in any patient’s DNA depending on the target size, but most of the variants are clinically irrelevant to the suspected diagnosis.

To aid in variant interpretation, Blueprint Genetics uses a comprehensive, proprietary variant database containing curated disease-related variants. The output of the bioinformatics pipeline is a comprehensive quality control report and annotated variant list for each sample including nomenclature, population frequencies, in silico predictions, literature and database (ClinVar, HGMD etc) hits and related links and previous interpretations (Internal + Databases) that all help the pathogenicity assessment.

A comprehensive clinical report

The final phase of Blueprint Genetics’ all-inclusive process is the geneticist’s statement, which is supported by the insight of a specialized clinician. For positive results, the report generally includes valuable insights into potential treatments, while highlighting any risks the disease presents to the patient. The statement is delivered to you through Nucleus, by mail, or by fax, depending on your preference.

If the results are negative, the statement helps move toward a potential future diagnosis by ruling out one or more possibilities. The geneticist’s statement may also include recommendations for a different, more comprehensive test when deemed beneficial. In this event, Blueprint Genetics generally does not require a new sample to run the recommended test, so it is easy for you to order if needed.